An epidermal growth factor receptor (EGFR) expression vector was introduced into an interleukin-3 (IL-3)-dependent myeloid call line, 32D, which is devoid of EGF receptors. EGFR expression allowed these cells to utilize EGF for signal transduction and continued proliferation. While 32D-EGFR cells remained nontumorigenic and factor-dependent on either IL-3 or EGFR transfection of v-erbB abrogated IL-3 dependence and induced tumorigenicity. These results establish that components of the EGF-signaling pathway necessary for transmitting intracellular signals must be available in these cells. The normal human EGFR expression vector was also transfected into NIH/3T3 cells to determine whether this growth factor receptor could induce transformation when overexpressed in NIH/3T3 cells. EGFR overexpression was shown to induce transformed foci or colonies in soft agar only after the addition of exogenous EGF. These findings indicate that overexpression of EGFR in human malignancies would confer a strong selective growth advantage to these cells in the presence of the ligand (EGF or transforming growth factor (TGF)- alpha). Cell lines transformed by H-ras or myc/raf-containing retroviruses were found to coexpress antigens usually restricted to the B-cell or myeloid pathways of differentiation. Detailed analyses of these lines suggested that the initial transforming event giving rise to these lines occurred in a precursor common to the B-cell and myeloid lineages. The availability of cell lines with both myeloid and B-cell differentiation potentials provides a unique opportunity to explore the molecular and biochemical events that define irrevocable commitment to these distinct hematopoietic lineages.